Worldwide Dissemination of blaKPC Gene by Novel Mobilization Platforms in Pseudomonas aeruginosa: A Systematic Review

The dissemination of blaKPC-harboring Pseudomonas aeruginosa (KPC-Pa) is considered a serious public health problem. This study provides an overview of the epidemiology of these isolates to try to elucidate novel mobilization platforms that could contribute to their worldwide spread. A systematic review in PubMed and EMBASE was performed to find articles published up to June 2022. In addition, a search algorithm using NCBI databases was developed to identify sequences that contain possible mobilization platforms. After that, the sequences were filtered and pair-aligned to describe the blaKPC genetic environment. We found 691 KPC-Pa isolates belonging to 41 different sequence types and recovered from 14 countries. Although the blaKPC gene is still mobilized by the transposon Tn4401, the non-Tn4401 elements (NTEKPC) were the most frequent. Our analysis allowed us to identify 25 different NTEKPC, mainly belonging to the NTEKPC-I, and a new type (proposed as IVa) was also observed. This is the first systematic review that consolidates information about the behavior of the blaKPC acquisition in P. aeruginosa and the genetic platforms implied in its successful worldwide spread. Our results show high NTEKPC prevalence in P. aeruginosa and an accelerated dynamic of unrelated clones. All information collected in this review was used to build an interactive online map.


Introduction
Hospital-acquired infections are considered one of the biggest challenges to patient safety [1]. In Europe, it is estimated that 2,609,911 new cases of healthcare-associated infections (HCAIs) are reported each year [2], and on average, around 80,000 hospitalized patients have at least one HCAI on any given day [3,4]. In the United States (USA), it is estimated that every day 1 in 31 hospitalized patients carry an HCAI [5]. Infections caused by Pseudomonas aeruginosa, which are resistant to antibiotics, have become a serious public health problem, representing a risk factor for hospitalized patients, especially those in intensive care units (ICUs) [6]. In 2017, multidrug-resistant (MDR) P. aeruginosa caused an estimated 32,600 infections among hospitalized patients and approximately 2700 deaths in the USA [7].
Regarding the genetic relation of the isolates, forty-one different sequence types (STs) were identified in 449 KPC-Pa in nine different countries ( Figure 2); the most predominant ST was ST463, to which 242 (53.9%) isolates belonged; other relevant STs were ST654, ST1212, ST664, and ST235 found in 32 (7.1%), 28 (6.2%), 21 (4.6%), and 20 (4.4%) isolates, respectively. In China, ST463 was the most frequent (68.9%) and exclusive to this country. Likewise, for ST654, 93.7% of the reports were part of the same study that evaluated isolates located in different hospitals in Argentina [61]. Although the pandemic clone ST235 does not have a high frequency, it has been described in at least five different countries. Furthermore, ST209 and ST274 reported in two studies conducted in Zhejiang, Figure 2. Geographic distribution of the blaKPC-harboring Pseudomonas aeruginosa isolates with known sequence types. The color shading represents the total number of isolates with known sequence types. Pie charts refer to the proportion of representative sequence blaKPC-harboring types found per country. Clonal complexes (CC) were assigned according to Del Barrio-Tofiño et al. in 2020 [73].

Genetic Platforms Mobilizing blaKPC Gene in P. aeruginosa
Out of all the isolates, only 234 KPC-Pa (36 studies) described the genetic location (plasmid or chromosome) and/or genetic structures surrounding blaKPC (transposons, NTEKPC, or insertion sequences). With regard to their genetic location, 199 (84.6%) of these isolates harbored blaKPC within a plasmid structure, 6 (2.5%) isolates contained the gene in the chromosome, and 1 isolate presented a report of blaKPC in the plasmid and chromosome. The remaining 28 (11.9%) KPC-Pa isolates did not emphasize the genetic location but did report the genetic environment adjacent to the blaKPC.
In the case of the seven reports harboring blaKPC on the chromosome, five (71.4%) of these reports were associated with an NTEKPC element and the other two (28.5%) with a Tn4401-like structure. Although the remaining 28 isolates did not specify the location of this gene on the plasmid or chromosome, 20 (71.4%) of them were associated with NTEKPC and 8 (28.5%) with Tn4401.

Genetic Platforms Mobilizing bla KPC Gene in P. aeruginosa
Out of all the isolates, only 234 KPC-Pa (36 studies) described the genetic location (plasmid or chromosome) and/or genetic structures surrounding bla KPC (transposons, NTE KPC, or insertion sequences). With regard to their genetic location, 199 (84.6%) of these isolates harbored bla KPC within a plasmid structure, 6 (2.5%) isolates contained the gene in the chromosome, and 1 isolate presented a report of bla KPC in the plasmid and chromosome. The remaining 28 (11.9%) KPC-Pa isolates did not emphasize the genetic location but did report the genetic environment adjacent to the bla KPC .
Of the 200 reports of bla KPC encoded within a plasmid, 52 (26.0%) reported the gene as part of an NTE KPC element, 44 (22.0%) in a Tn4401 transposon variant, and 1 isolate (0.5%) reported bla KPC within an integron-like genetic structure [66]. KPC-associated plasmids varied widely in size, from small plasmids of less than 4 Kbp to mega plasmids greater than 400 Kbp [19,44]. Furthermore, it was possible to identify seven different incompatibility groups: IncP [50], IncP-3-like (IncA/C) [19], IncU [27,49], IncP-6 [17,27,38], IncF-like [66], IncQ1 [21], and IncHI1 [18]. According to the information collected, only 24 bla KPC -carrying P. aeruginosa plasmids were fully sequenced and are available both in the NCBI public database and the published literature (Table 2).  In the case of the seven reports harboring bla KPC on the chromosome, five (71.4%) of these reports were associated with an NTE KPC element and the other two (28.5%) with a Tn4401-like structure. Although the remaining 28 isolates did not specify the location of this gene on the plasmid or chromosome, 20 (71.4%) of them were associated with NTE KPC and 8 (28.5%) with Tn4401.

In Silico Assessment of the bla KPC Genetic Environment on P. aeruginosa Isolates
Due to the high presence of possible NTE KPC in these isolates, an analysis to characterize the bla KPC genetic environment was performed. The genetic environments of the isolates that were collected from the NCBI nucleotide database were compared to establish their relationship. Since Chen et al. coined the term NTE KPC in 2014 for non-Tn4401 elements adjacent to the bla KPC found mainly in K. pneumoniae [24], three types (I, II, and III) and several subtypes have been reported. Currently, when a new structure is found, the assignment of type and subtype relies on the author. This has generated some problems such as the duplication of names in different structures; for instance, two of these elements that were reported in different species were both named as NTE KPC -IIe despite presenting structural differences between them; specifically, the bla TEM region that both environments presented is not shared. Although both present a tnpA of the Tn3 family upstream of the resolvase, they do not seem to have similarities in most of their structure. In this sense, to unify the nomenclature, we have renamed some structures according to their order of appearance. Thus, the NTE KPC -IIe that was reported by Campana [74] was renamed NTE KPC -IIf, and the structure reported by Abril et al. (NCBI accession number CP095773.1) as NTE KPC -IIf has received the name NTE KPC -IIg ( Figure 3). From our results, 25 different and novel environments were found in GenBank, which have not previously been reported as NTE KPC . Supplementary material (Table S1) shows the list of isolates harboring novel NTE KPC structures. Among these, seventeen (68.0%) were reported as belonging to subtype I, six to subtype II (24.0%), one to subtype III (4.0%), and one that could not be classified within the three existing subtypes of NTE KPC (4.0%) (pCCBH8525_KPC). The bla KPC gene found within the plasmid pCCBH28525_KPC (Table 2) did not present any of the surrounding characteristic genes of the NTE KPC subtypes already described: ISKpn27 in the case of NTE KPC -I; bla TEM for NTE KPC -II; and IS6100/Tn5563 resolvase for NTE KPC -III [24]. Instead, the genes encoding hypothetical proteins were located in the upstream region, and genes related to transposition, such as ISKpn6, tnpA (encoding a Tn3-related transposase), and tnpR, were found downstream ( Figure 4). For this reason, we considered that this structure corresponds to a new type, namely NTE KPC -IVa (CP086065.1). It should be clarified that in the present study, the frequency of these novel genetic environments was not evaluated as the intention was to report the differences among novel mobilization platforms found in P. aeruginosa compared to those previously reported [24].
On the other hand, a large part of the 25 novel environments that were classified as NTE KPC -I was subclassified as NTE KPC -Ib-like elements as they contain the highly conserved region made up of an incomplete version of the ISKpn6, korC, klcA, and repA genes downstream to bla KPC . In the upstream region, they contained ISKpn27 and, in many cases, a resolvase (tnpR). They also presented different transposable elements, such as the tnpR of Tn4653, Tn1721, and Tn2 transposases, or different copies of the IS26, depending on the isolate ( Figure 5). NTE KPC -I was the only type found to be chromosomally located with subtle structure variations. For instance, the region surrounding chromosomal bla KPC in isolate SRRSH1521 (ST244) (CP077997.1) showed 97.2% homology to a region covering 87% of the NTE KPC -Ib gene, with the major difference being the insertion of an IS6100 copy (instead of IS26) into the tnpR gene. In addition, an NTE KPC -Ic-like element was also identified in the isolate BH6 (CM003767.1) but with some deletions upstream and downstream of the bla KPC gene ( Figure 5).   (Table S1) shows the list of isolates harboring novel NTEKPC structures. Among these, seventeen (68.0%) were reported as belonging to subtype I, six to subtype II (24.0%), one to subtype III (4.0%), and one that could not be classified within the three existing subtypes of NTEKPC  Table S1. teins were located in the upstream region, and genes related to transposition, such as IS-Kpn6, tnpA (encoding a Tn3-related transposase), and tnpR, were found downstream (Figure 4). For this reason, we considered that this structure corresponds to a new type, namely NTEKPC-IVa (CP086065.1). It should be clarified that in the present study, the frequency of these novel genetic environments was not evaluated as the intention was to report the differences among novel mobilization platforms found in P. aeruginosa compared to those previously reported [24]. On the other hand, a large part of the 25 novel environments that were classified as NTEKPC-I was subclassified as NTEKPC-Ib-like elements as they contain the highly conserved region made up of an incomplete version of the ISKpn6, korC, klcA, and repA genes downstream to blaKPC. In the upstream region, they contained ISKpn27 and, in many cases, a resolvase (tnpR). They also presented different transposable elements, such as the tnpR of Tn4653, Tn1721, and Tn2 transposases, or different copies of the IS26, depending on the isolate ( Figure 5). An interesting and complex chromosomal NTE KPC structure was characterized for isolate NDTH9845, where three copies of bla KPC were mobilized through very similar NTE KPC -Ib platforms. Some differences among these three platforms were the insertion of different IS30 copies and the Tn2-like tnpA gene ( Figure 6).
Among the six isolates that contained structures related to NTE KPC -II, five were considered NTE KPC -IIa-like elements, presenting some structural differences, such as the deletion of ISKpn27 (i.e., isolate pFAHZU40-KPC); insertion of transposable elements, such as the IS26, ISApu2, ISApu1, Tn3, and Tn2-like transposases; or changes in the intergenic regions ( Figure 7). Interestingly, the latest NTE KPC -II described corresponds to the recently described NTE KPC -IIg, which was also found in different clones recovered from Colombia and Argentina, which are two distant countries, suggesting a wide dissemination of this platform (access through CP095773.1 and OL780449.1, respectively).
Finally, only one isolate presented an NTE KPC -III environment. This case is particular as it harbors IS6100 (a hallmark of the NTE KPC -III) in the form of a chimera with a previously unreported transposase. This transposase shows similarities with those of the Tn3 or IS481 family. In addition, genes normally found in other NTE KPC subtypes, such as types I or II, are found downstream and correspond to korC, klcA, and repB.

Interactive Online Map Construction
The data from this systematic review show only an approximation of the current landscape of bla KPC -harboring CRPA. The SARS-CoV-2 pandemic taught us the importance of generating interactive tools to collect worldwide information about infectious pathogens in real time. Based on this, we summarized the valuable information presented here on bla KPC acquisition and spread in P. aeruginosa in a world online map tool, which can be consulted at the following link: https://maphub.net/LGMB/KPC-Pseudomonas-aeruginosa-LGMB. The information contained in this map will be updated periodically with new genomic reports of KPC-Pa isolates.  (ST244) (CP077997.1) showed 97.2% homology to a region covering 87% of the NTEKPC-Ib gene, with the major difference being the insertion of an IS6100 copy (instead of IS26) into the tnpR gene. In addition, an NTEKPC-Ic-like element was also identified in the isolate BH6 (CM003767.1) but with some deletions upstream and downstream of the blaKPC gene (Figure 5). An interesting and complex chromosomal NTEKPC structure was characterized for isolate NDTH9845, where three copies of blaKPC were mobilized through very similar NTEKPC-Ib platforms. Some differences among these three platforms were the insertion of different IS30 copies and the Tn2-like tnpA gene ( Figure 6). Among the six isolates that contained structures related to NTEKPC-II, five were considered NTEKPC-IIa-like elements, presenting some structural differences, such as the deletion of ISKpn27 (i.e., isolate pFAHZU40-KPC); insertion of transposable elements, such as the IS26, ISApu2, ISApu1, Tn3, and Tn2-like transposases; or changes in the intergenic regions ( Figure 7). Interestingly, the latest NTEKPC-II described corresponds to the recently described NTEKPC-IIg, which was also found in different clones recovered from Colombia and Argentina, which are two distant countries, suggesting a wide dissemination of this platform (access through CP095773.1 and OL780449.1, respectively).
Finally, only one isolate presented an NTEKPC-III environment. This case is particular as it harbors IS6100 (a hallmark of the NTEKPC-III) in the form of a chimera with a previously unreported transposase. This transposase shows similarities with those of the Tn3 or IS481 family. In addition, genes normally found in other NTEKPC subtypes, such as types I or II, are found downstream and correspond to korC, klcA, and repB.

Interactive Online Map Construction
The data from this systematic review show only an approximation of the current landscape of blaKPC-harboring CRPA. The SARS-CoV-2 pandemic taught us the importance of generating interactive tools to collect worldwide information about infectious pathogens in real time. Based on this, we summarized the valuable information presented here on blaKPC acquisition and spread in P. aeruginosa in a world online map tool, which can be consulted at the following link: https://maphub.net/LGMB/KPC-Pseudomonas-aeruginosa-LGMB. The information contained in this map will be updated periodically with new genomic reports of KPC-Pa isolates.

Discussion
This study systematically reviewed fifty-three studies and provides a first insight into the impacts of genetic platforms on the dissemination of bla KPC in P. aeruginosa and shows an overview of the global epidemiology of bla KPC -harboring CRPA. Since the first report of KPC-Pa in 2007 [22], the number of these bla KPC -harboring isolates has been increasing, especially in Asia and South America; specifically, China, Brazil, Colombia, and Puerto Rico are the countries with the largest number of reports on the subject. According to the Antimicrobial Testing Leadership and Surveillance program [75], the rate of resistance to carbapenems (reported in 2018) in P. aeruginosa clinical strains was highest in the Middle East, followed by South America, Europe, and North America [13], suggesting that KPC may potentially be contributing to the increase and spread of these patterns of resistance in conjunction with other carbapenemases (i.e., VIM) and resistance mechanisms such as the repression of the OprD porin and the overexpression of efflux pumps [15,28,76].
The WHO classified CRPA as a critical priority on the list of the most dangerous pathogens that need the development of new antimicrobial drugs [77]. Even though bla KPC is still most prevalent in K. pneumoniae [18], our findings suggest it may also play a role in the ability of P. aeruginosa to exhibit genome plasticity and adapt to different conditions across 14 countries [13].
The current systematic review indicated that KPC-2 remains the most successful KPC variant worldwide, followed by KPC-5, which has only been reported in Puerto Rico. Recently, KPC-33 and KPC-90 variants have also been identified in Zhejiang, China, both belonging to ST463 [36,41]. Although all isolates have reported resistance against carbapenem antibiotics, some variants, such as KPC-90, have already shown to be resistant to more advanced combinations of antibiotics, such as ceftazidime-avibactam (CZA), a treatment that has demonstrated high efficacy against bla KPC-2 -harboring CRPA strains [41].
In addition, there is an isolate that was recently identified by us (not yet published), which was not part of the review. This isolate apparently corresponds to the first report of bla KPC-3 in P. aeruginosa, is associated with Tn4401b, and was found in the pandemic highrisk clone ST111. The appearance of the second most widespread KPC-3 variant [78] within a highly active transposon mainly responsible for KPC dissemination in carbapenemresistant Enterobacteriaceae [24] opens a possible new expansion route for this enzyme. Furthermore, since the completion of this review, there have been reports of KPC-3 variants, such as KPC-31, which are also resistant to CZA and were found in a high-risk clone ST235 [79]. These findings underline the importance of continued surveillance.
We have identified more than forty-one different STs, of which only two (ST209, ST274) presented an SLV. This finding demonstrates a wide heterogeneous distribution of non-related clones. These SLVs will diversify over time, generating new variants, likely including double locus variants (DLV) and triple locus variants (TLV) [80]. Additionally, seven out of ten high-risk clones reported for P. aeruginosa (ST111, ST244, ST235, ST277, ST308, ST357, and ST654) were identified [13], and the ST that showed the greatest spread was ST235, following reports in Argentina, Brazil, Colombia, Germany, and Nepal. Our results are consistent with previous studies that describe the population structure of P. aeruginosa as non-clonal and epidemic [81]. However, it was observed that the ST463 reports correspond mostly to hospitals located in East China, which could represent endemicity for this clone [36]. Likewise, it has been shown that the high-risk clone ST654 is playing a key role in the spread of KPC-Pa in Argentina [61] as opposed to different clones being responsible for its spread in Colombia.
The global expansion of carbapenem resistance in Gram-negative bacteria can be attributed to horizontal gene transfer mediated by active transposons and multiple plasmids [36]. This has allowed enzymes, such as KPC, to be more easily transferred and become endemic in various places, such as the USA, Argentina, Brazil, Colombia, Eastern China, Greece, Israel, and Italy [24,36]. The worldwide distribution of bla KPC in K. pneumoniae has been attributed mainly to two factors: (i) the global dissemination of the clonal group CG258 and (ii) the localization of bla KPC in the Tn4401 transposon variants harbored in different plasmids [60,82]. Nonetheless, for P. aeruginosa, we observed an alarming increase in unrelated clones and the localization of bla KPC within NTE KPC structures. In this review, we continue to observe a high incidence of Tn4401 elements, among which their isoform b stands out, but Tn4401a is also reported. Likewise, it has been shown that the wide circulation of plasmids carrying bla KPC has resulted in an increase in resistant isolates reported in hospitals. Up to now, twenty-four bla KPC -carrying plasmids from P. aeruginosa have been available in the NCBI public databases and the published literature. It would be interesting to understand the possible associations between plasmids and mobilization platforms, which may be the subject of future studies.
On the other hand, the bla KPC gene has been reported within these conventional genetic elements in several bacterial species, including P. aeruginosa [24]. The presence of NTE KPC in the KPC-Pa isolates identified in this review shows that these elements may be contributing to the dissemination of KPC. In addition, our in silico analysis shows a great diversity of NTEs, with NTE KPC -I being the most prevalent. However, in some cases, the NTE KPC -Ib-like elements were present as several copies, resulting in chimeras with various copies of bla KPC . There was a case (chromosome of NDTH7329 strain) in which six identical copies of the same genetic environment were present (accession number CP078006.1). The presence of transposases, such as IS26 and Tn3 family transposases, including Tn3 itself, Tn2 or TnAs1, and TnA2, may suggest that the mobilization of the NTE KPC -I elements through the Russian doll model in which transposable elements are mobilized through adjacent transposable elements [83]. The frequency of IS26 in the isolates evaluated by this study is a particularly interesting result because IS26 can be mobilized in various ways; in fact, it is believed that it could mobilize neighboring genes without the action of another transposase [84].
It is noteworthy that there were isolates in which the same mobilization platform was present, as in the case of the mobilization platform found in the SRRSH1521 (CP077997.1) chromosome, where: (i) theYLH6_p3 plasmid mobilization platform was also present in the SRRH1042 plasmid, and (ii) the mobilization elements in pZYPA01 were also present in pP23-KPC. This suggests that these elements are able to transfer between plasmids, at least within the Chinese territory where these cases were reported. Despite the high incidence in NTE KPC reports, it is still not clear whether these genetic structures can be mobilized independently or whether their successful dissemination is due to the involvement of various plasmids, so further analysis of these platforms is warranted. The NTE KPC -IIe is the only element of this kind that has shown defined inverted repeats (IRs) in addition to presenting direct repeats as an indication of its transposition [85].
The present systematic review encountered the following limitations: Due to the large amount of information related to CRPA strains, our search strategy was focused on mobilization platforms; this could exclude relevant information from studies with a different approach. In addition, although duplicate isolates were not considered for analysis in this study, there are studies that reported multiple isolates possibly associated with epidemic outbreaks or surveillance studies, which causes an over-representation in some of the data presented in the review. However, as they are independent isolates, it was decided to evaluate all the information to avoid losing relevant data, such as STs. Finally, most of the evaluated data in this review required bioinformatics information that many of the articles do not present.

Search Strategy
A systematic review was conducted following the published guidelines for the development of systematic reviews [86]. Online searches were performed through the PubMed and EMBASE databases for articles published up to June 2022, without geographic location restriction. A combination of keywords, controlled vocabulary (MeSH/Emtree terms), and Boolean operators ("AND" and "OR") were used during the search. The initial search strategy was designed using a fitted PICO (population, intervention, comparison, outcome) model and combining terms related to the pathogen ("Pseudomonas aeruginosa"), carbapenem antibiotics ("carbapenem", "doripenem", "ertapenem", "imipenem"), the resistance gene bla KPC ("beta-lactamase KPC", "bla KPC ", "KPC"), and some dissemination platforms or MGE ("dissemination", "transposon", "plasmids", "DNA transposable elements") that could be associated with the spread of these strains worldwide. Supplementary Material Table S2 shows the full search strategies for each database. Additionally, via hand-searching, we complemented the search to include relevant articles that were missed during indexing.
The initial protocol was registered in the Prospective International Registry of Systematic Reviews (PROSPERO) of the National Institute of Health Research (Registration code: CRD42022320686).

Inclusion and Exclusion Criteria
Full-text retrieved items were screened to determine their eligibility according to the predefined selection criteria. Based on the objective, all the observational studies related to KPC-Pa isolates from clinical samples of patients with resistance to at least one type of carbapenem antibiotic were considered for inclusion in the systematic review. Furthermore, studies that reported dissemination platforms or MGE related to bla KPC and/or studies that reported STs associated with the isolates were included. Furthermore, we included some articles that did not describe the dissemination platforms but were considered relevant as they provided information related to the emergence of CRPA in new geographical locations.
We excluded studies with isolates of CRPA from environmental or animal samples, non-CRPA, other Pseudomonas spp., and studies solely reporting P. aeruginosa without KPC. Reviews (systematic, meta-analysis, and narrative), editorials, conferences, meeting abstracts, and duplicate reports were excluded. Articles in languages other than English and Spanish, with missing information and without full text, were not considered. Supplementary material (Table S3) shows the list of excluded studies with reasons for exclusion.
The quality of the studies was not considered an exclusion criterion for this systematic review.

Data Extraction and Analysis
The titles and abstracts of the literature search results were screened for eligibility and annotated in our database by one study researcher (D.F-H) following the PICO-based, predefined selection criteria.
The following data were extracted from each study according to inclusion criteria: (i) study-related variables (the first author's name, year of publication, and country where the study was conducted), (ii) isolate-related variables (collection date, strain name, KPC variant, and STs), and (iii) genetic location (plasmid or chromosome) and genetic structures surrounding bla KPC (transposons, NTE KPC , and/or insertion sequences). Information not provided by the article was classified as "not specified". We performed typing of the plasmids that had an available accession number and did not report an incompatibility group using PlasmidFinder [87]. Additionally, an SLV analysis was conducted using PubMLST and eBURST tools [80,88]. The statistical analysis of the data obtained in this systematic review was performed using the package SPSS ® . Finally, we provided a narrative synthesis of the results from the studies included, structured around the geographic distribution of bla KPC reports and possible mobilization platforms.

Exploration of the bla KPC Genetic Environment for P. aeruginosa in GenBank
A database was created for compiling information on the bla KPC genetic environments in P. aeruginosa collected in GenBank (up to June 2022). All partial or fully sequenced nucleotide entries with more than 3000 bp upstream bla KPC were included. General information of the entries, such as country, length, replicon type (linear or circular), bla KPC variant, position in the genome, isolate name, and access information (GenBank and PMCID access numbers) were also registered. The nucleotide sequence for all entries was exported and compared against reference sequences of the NTE KPC different subgroups (I, II, and III) previously described, whose classification criteria are based on the region upstream of bla KPC , as described by Chen et al. in 2014 [24]. The criteria to classify NTE KPC -I was the presence of ISKpn27 or the characteristic tnpR of this subtype of elements; for NTE KPC -II, the insertion of ∆bla TEM ; and for NTE KPC -III, the insertion of Tn5563/IS6100 [24].
In the case of no association with previously reported genetic environments, the entry was characterized by manual curation using Artemis Comparison Tool (ACT), BLASTn and BLASTp [89,90], and specialized databases for MGE (TnRegistry and ISFinder) and resistance genes (CARD) [91,92]. Paired alignments were developed and plotted using Easyfig [93], showing identity between pairs in a window of 300 bp; the isolates in figures were organized according to the percentage of identity obtained from previously paired alignments of all the sequences in BLASTn.

Participation of Patients in the Study
The patients mentioned in this study were not involved in conducting or reporting plans for this systematic review.